Platelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events [1]. Platelets promote both thrombus formation and the release of inflammatory markers, thus, previous studies focused on a possible correlation between platelet count and blood levels of inflammation and haemostasis biomarkers and its cardiovascular risk [2], [3], [4]. Previously, platelet activation has been described to be associated with thromboischemic events following coronary interventions and stenting [5], [6]. Platelet activation markers such as P-selectin (CD62P) and platelet glycoprotein VI (GPVI) may provide a predictive and prognostic value, thus, an increased platelet activation is associated with a poor clinical outcome in patients following acute myocardial infarction and ischemic stroke [7], [8], [9], [10]. However, CD62P is found in both platelets as well as in endothelial cells in which it is stored in thrombocytic granules and Weibel-Palade bodies, respectively, whereas platelet GPVI has an improved stamina in circulation, is limited to the megakaryocyte system and, thus, is platelet specific [11]. Previously, we showed that changes in platelet GPVI expression may indicate an imminent myocardial infarction before rise of biomarkers of myocardial necrosis such as troponin and creatinkinase occurs when already myocardial ischemia occurred [7], [8].

The aim of the present study was to evaluate the influence of platelet count on the surface expression of platelet GPVI in patients with symptomatic coronary artery disease, namely stable angina pectoris (SAP) or ACS respectively. Both, platelet count and platelet GPVI surface expression may be useful as potential markers for an ACS.

We consecutively evaluated 414 patients presented with SAP and 429 patients with ACS. All patients underwent coronary angiography and blood was sampled used for the flow cytometric analysis of surface expression of platelet GPVI. All of the subjects had a mean age of 66.5Â±13.1 years. The exclusion criteria were age below 18 years, lack of informed consent, any history of malignancy, connective tissue disease, thyroid disease, renal disease, use of steroids, hormone replacement therapy, patients with nondiagnostic electrocardiogram. Clinical outcome was assessed by telephone interview after a follow-up at three months or by hospital chart analysis in 806 patients (95.6%).

The surface expression of the platelet receptors GPVI and CD62P was determined by two-color whole-blood flow cytometry, as described previously [12]. Each measurement was performed in duplicate and mean fluorescence intensity (MFI) was used as the index of receptor expression. Fluorescein (FITC)-conjugated anti-CD62P (clone CLB-Thromb/6) and phycoerythrin (PE)-conjucated anti-CD42b (clone SZ2) monoclonal antibodies (mAb) were purchased from Immunotec, Beckman Coulter, Inc., USA. The FITC-conjugated anti-GPVI mAb 4C9 was generated and characterized as previously described [13], [14]. Plasma levels of soluble GPVI were determined through a self-developed enzyme-linked immunosorbent assay kit using the anti-GPVI mAb [14]. The detection range of soluble GPVI was 0.08–5 ng/mL.

We found 228 (27.1%) females and 615 (72.9%) males, 634 (75.2%) with arterial hypertension, 473 (56.1%) with hyperlipidemia, 295 (34.9%) with diabetes mellitus, 161 (19.1%) with a family history of coronary artery disease, and 308 (36.5%) smoking.

To test whether platelet count is influenced by confounders, comparison of the decadic logarithm of platelet count between ACS and SAP was adjusted by possible confounders such as medical treatment, cardiovascular risk factors, gender, age, left ventricular ejection fraction, and degree of coronary artery disease. Apart from age (p=0.004) and gender (p=0.024), multifactorial analysis of covariance revealed that the decadic logarithm of platelet count for ACS is independent of confounders.

Patients in the first quartile tended to have a poorer clinical composite outcome (myocardial reinfarction, ischemic stroke, cardiovascular death). Considering cardiovascular death, the second quartile with a platelet count of 208–246x109/L tended to have the lowest mortality represented in the Kaplan-Meier survival analysis (Figure 2 [Fig.Â 2]). However, the results did not reach a significant level (Log rank: p=0.083).

The findings of this observational study are that patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count, which was paralleled in trend for CD62P expression and in a small subgroup of patients with ACS for soluble GPVI. According to platelet count, patients in the first quartile tended to have a poorer clinical composite outcome (myocardial reinfarction, ischemic stroke, cardiovascular death). Considering cardiovascular death, the second quartile of platelet count tended to have the lowest mortality represented in the Kaplan-Meier survival analysis.

Intriguingly, we have seen that ACS patients with high platelet count presented with fewer activated platelets, therefore, it appears to be a down-regulation of platelet GPVI in ACS patients with high platelet count. In contrast, platelet count in patients with stable angina did not show any significant difference for GPVI and CD62P expression on lower expression levels than in patients with ACS [7], [8]. Although platelet GPVI is constitutively surface-expressed, platelet activation usually leads to further release and enhanced plasma membrane expression of GPVI as shown in patients with ACS and ischemic stroke [7], [8], [9]. However, recent experimental studies also found that platelet GPVI can be down-regulated in vivo using murine models [15], [16], [17]. The process of down-regulation may occur through two distinct pathways, namely ectodomain shedding or internalization/intracellular clearing [16]. Particularly, the metalloproteinase-dependent shedding is considered as a crucial effect for the down-regulation process of GPVI. Thus, platelet activation induces metalloproteinase-dependent GPVI cleavage to down-regulate platelet reactivity to collagen [17]. To support this pathophysiologic construct, we determined soluble GPVI in plasma showing similar results to the GPVI platelet surface expression. On the other hand, soluble GPVI may also act as an inhibitor of atherothrombosis [18]. Therefore, the level of soluble GPVI reflects the activation of circulating platelets.

Our results with the second quartile of platelet count showing the lowest mortality in three-months follow-up were paralleled by recent findings on platelet count in patients with unstable angina or non-ST elevation myocardial infarction for long-term mortality [19]. Other studies found contrasting results identifying low platelet count as an independent risk factor for mortality at 30 days [20], whereas initially increased platelet count in ACS may predict infarct artery flow suggesting the intensified use of antiplatelet drugs [21].

In conclusion, A lower platelet count is associated with higher platelet GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased platelet activation and enhanced recruitment of platelets to the vascular lesion.

Further studies should examine whether both platelet count and determination of initial platelet activation markers might be a prognostic tool for thromboischemic events such as ACS and ischemic stroke.